1. Field of the Invention
This invention relates to novel labeled conjugates for use in specific binding assays for ligands or their binding partners in a liquid medium. In particular, the invention relates to flavin adenine dinucleotide (FAD)-labeled conjugates for use in such assays, particularly for determining an iodothyronine such as thyroxine in serum. The invention further relates to intermediate compounds produced in the synthesis of the novel labeled conjugates.
The iodothyronines have the following general formula: ##STR2## wherein .beta..sup.1 and .beta..sup.2 are, independently, hydrogen or iodine. The principal iodothyronines of clinical interest are listed in Table 1 below.
TABLE 1 ______________________________________ Iodothyronine .beta..sup.1 .beta..sup.2 ______________________________________ 3,5,3'5'-tetraiodothyronine iodine iodine (thyroxine; T-4) 3,5,3'-triiodothyronine iodine hydrogen (liothyronine; T-3) 3,3',5'-triiiodothyronine hydrogen iodine ("reverse" T-3) 3,3'-diiodothyronine hydrogen hydrogen ______________________________________
The quantitative determination of the concentration of the various iodothyronines, particularly the hormones T-3 and T-4, in serum and of the degree of saturation of the iodothyronine binding sites on the carrier protein thyroid binding globulin (TBG) are valuable aids in the diagnosis of thyroid disorders. Likewise, the determination of other components of body fluids including serum is useful in assessing the well-being of an individual. Examples of other substances of clinical interest are evident from the description below.
2. Brief Description of the Prior Art
Specific binding assay methods have undergone a technological evolution from the original competitive binding radioimmunoassay (RIA) in which a radioisotope-labeled antigen is made to compete with antigen from a test sample for binding to specific antibody. In the RIA technique, sample antigen is quantitated by measuring the proportion of radioactivity which becomes associated with the antibody by binding of the radiolabeled antigen (the bound-species of the labeled antigen) to the radioactivity that remains unassociated from antibody (the free-species) and then comparing that proportion to a standard curve. A comprehensive review of the RIA technique is provided by Skelly et al, Clin. Chem. 19: 146(1973). While by definition RIA is based on the binding of specific antibody with an antigen or hapten, radiolabeled binding assays have been developed based on other specific binding interactions, such as between hormones and their binding proteins.
From the radiolabeled binding assays have evolved nonradioisotopic binding assays employing labeling substances such as enzymes as described in U.S. Pat. Nos. 3,654,090 and 3,817,837. Recently further improved nonradioisotopic binding assays have been developed as described in German Offenlegungschriften Nos. 2,618,419 and 2,618,511, based on U.S. Ser. Nos. 667,982 and 667,996, filed on Mar. 18, 1976 and assigned to the present assignee, employing particularly unique labeling substances, including coenzymes, cyclic reactants, cleavable fluorescent enzyme substrates, and chemiluminescent molecules. Flavin adenine dinucleotide is mentioned as being useful as a coenzyme label since FAD functions as a coenzyme in useful monitoring reactions. In U.S. Patent application Ser. No. 917,961, filed June 22, 1978 and assigned to the present assignee, FAD is further described as useful in improved specific binding assays employing a prosthetic group as the label because FAD also functions as a prosthetic group in select biochemical systems.
Various methodologies exist for the determination of iodothyronine concentrations in serum. A significant advance in iodothyronine assays was the development of the competitive protein binding assay by Murphy and Pattee, J. Clin. Endocrinol. Metab. 24:187(1964) in which radiolabeled iodothyronine competes with serum iodothyronine for binding to TBG. The development of specific antiserum for the various iodothyronines permitted radioimmunoassays to be devised in which radiolabeled and serum iodothyronine compete for binding to antibodies rather than to TBG. In both the competitive protein binding assay and the radioimmunoassay for an iodothyronine, the radiolabeled material consists of the native iodothyronine in which one or more of the iodine atoms are replaced by a radioactive iodine isotope, usually .sup.125 I. The above-mentioned nonradioisotopic binding assays have offered even more advantageous methods for determining iodothyronines, particularly those methods described in U.S. Pat. Nos. 4,043,872 and 4,040,907 and most especially in OLS's 2,618,419 and 2,618,511 and U.S. Ser. No. 917,961 mentioned above.